US4810656A - Chromogenic reactive for determining the iron content and the iron-binding capacity of biological liquids - Google Patents

Chromogenic reactive for determining the iron content and the iron-binding capacity of biological liquids Download PDF

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Publication number
US4810656A
US4810656A US06/947,133 US94713386A US4810656A US 4810656 A US4810656 A US 4810656A US 94713386 A US94713386 A US 94713386A US 4810656 A US4810656 A US 4810656A
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United States
Prior art keywords
reagent
iron
concentration
binding capacity
active substance
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US06/947,133
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English (en)
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Giorgio Torelli
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CHEMICAL LABORATORIES SR A CORP OF ITALY
Chemical Laboratories Srl
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Chemical Laboratories Srl
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Assigned to CHEMICAL LABORATORIES S.R. A CORP. OF ITALY reassignment CHEMICAL LABORATORIES S.R. A CORP. OF ITALY ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: TORELLI, GIORGIO
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/91—Iron-binding capacity of blood
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • the present invention relates to an improved reactive for determining the iron content and the overall iron-binding capacity of biological liquids, in particular of serum.
  • this reagent was also not free from the proteic and ionic interferences; the increase in color due to the interfering substances was simply compensated by the inability to extract and meter all the iron present in the serum.
  • the object of the present invention is to provide an improved chromogenic reagent for determining the iron content and the iron-binding capacity of biological liquids, in particular of serum, suitable to eliminate the disadvantages of the similar reactives currently in use.
  • the specific aim of the present invention is to provide a chromogenic reagent as described above which eliminates the ionic and/or proteic interfering agents of the serum in the iron-chromogen reaction of the reagent.
  • a chromogenic reagent for determining the iron content and the iron-binding capacity of serum comprising a chromogen composed of chromazurol B (CAB), a surface-active substance and a buffer for keeping the pH of the reactive at 4.5-5, characterized in that it comprises at least one salt in an amount suitable to impart to the reagent an ionic strength, expressed in terms of NaCl concentration, of at least 100 g/l.
  • CAB chromazurol B
  • the same comprises said surface-active substance in a concentration higher than 500 mg/l.
  • the reagent according to the present invention jointly comprises said surface-active substance in a concentration of at least 500 mg/l, at least one salt for an ionic strength expressed in terms of NaCl concentration of at least 100 g/l and furthermore comprises at least one aminoacid or a derivative thereof suitable for selectively binding the Cu 2+ in an amount equal to a glycine concentration ranging between 1 and 40 g/l.
  • FIG. 1 is a colorimetric absorption spectra of a serum sample to which the chromogenic reagent of the present invention has been added and of a control sample;
  • FIGS. 2 and 3 illustrate absorption spectra of chromogenic reagents reacted with interfering proteins
  • FIG. 4 illustrates absorption spectra on a standard (St), on a control serum (Rn) and on two human test sera (S 1 and S 2 ).
  • FIG. 1 illustrates the colorimetric absorption spectrum of CAB to which an aminoacid has been added, according to the present invention, and in contact with a serum containing copper ions (lower curve) in comparison with the spectrum of the CAB without aminoacid in contact with an identical serum containing the above said ions (upper curve).
  • FIG. 2 illustrates absorption spectra of chromogenic reagents which have reacted with interfering proteins, of which the continuous curve relates to the reagent according to the present invention, the dot-and-dash curve relates to a reagent with a halved concentration of surface-active substance, the broken line curve relates to a reagent with the ionic strength reduced to one third and the dotted curve relates to a reagent without surface-active substance.
  • proteic interferences examined in this figure have been studied by precipitating in the serum, with EDTA (100-150 mg/ml) the ions (among which iron, too) which react with the chromogen.
  • FIG. 3 again illustrates absorption spectra of reagents which have reacted with interfering proteins, of which the continuous curve is related to the reagent according to the invention, the dot-and-dash curve relates to the same reagent with a halved concentration of surface-active substance, the broken line curve relates to the reagent according to the invention but with the ionic strength reduced to one third and the dotted curve relates to the same reagent without CAB.
  • the originality of the research which has led to the present invention lies in that it has shown that, by significantly increasing the concentration of the surface-active substance, the protein-CAB interactions decrease.
  • the spectrum on the standard has the same pattern as the spectrum on the sera, and therefore the calculated values of iron concentration remain constant as ⁇ varies.
  • FIG. 4 depicts, to the left, the absorption spectra on a standard (St) (consisting of a Fe 3+ 30 Mmol/l solution), on a control serum (Rn) (consisting of Control serum Roche) and on two human test sera (S 1 and S 2 ).
  • St standard
  • Rn control serum
  • S 1 and S 2 human test sera
  • the chromogen used in the reagent according to the present invention is ex conc. BN azure dichrome (C.I. mordant blue 1/4380) commonly termed chromazurol B (CAB).
  • a quantity of chromogen is used which may vary between 40 to 80 mg per liter of reagent.
  • the surface-active substance which can be used in the reagent according to the invention may be any one commonly used in similar analytical reagents.
  • CTMA cetyltrimethylammonium bromide
  • Triton X100 produced by B. D. H. and composed of isooctylphenoxypolyethoxyethanol
  • the salt which may be used to impart the ionic strength required in the reagent according to the invention may be any. Obviously, for reasons of availability and cost, NaCl is preferred.
  • the ionic strength of the reagent is calculated, as is known, according to the formula ##EQU2##
  • the aminoacid preferably used in the reagent according to the invention is glycine or its derivative glycylglycine, glycylalanine, but it is equally possible to use other aminoacids such as alanine, glutamic acid, etc., or a derivative thereof containing said aminoacids in its structure.
  • the reagent according to the invention may be prepared by means of conventional methods by purifying CAB, salifying it with the amount of salt suitable to impart the abovementioned ionic strength thereto, and mixing it with the surface-active substance, the aminoacid and an appropriate buffer suitable to keep the pH at 4.5-5, e.g. a buffer of acetic acid-sodium acetate.
  • the following example illustrates a method of preparing the reagent according to the invention.
  • the reagent thus obtained is useful in determining the concentration of iron and the iron-binding capacity of serum with the conventional colorimetric methods, without the use of blank sample.
  • the sample of serum, a standard and a blank composed of distilled water are mixed with an equal amount of reagent, and after accurate agitation of the mixture the readout is performed of the absorption of the sample and of the standard against the blank, at 630 nm. Then the iron concentration is calculated with the formula described above in relation to FIG. 4.
  • the sample of serum is saturated with an iron saturation agent, then left to react after accurate agitation, the excess iron is removed by absorption on carbon dust and subsequent centrifugation and the iron present in the supernatant is metered with the same process used above to determine the iron.
  • the reagent according to the invention achieves the intended objects, constituting a safe and reliable means of analysis which eliminates the ionic and proteic interferences and allows to achieve accurate and reproducible analysis results.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US06/947,133 1985-12-27 1986-12-29 Chromogenic reactive for determining the iron content and the iron-binding capacity of biological liquids Expired - Fee Related US4810656A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT23396/85A IT1201512B (it) 1985-12-27 1985-12-27 Reattivo cromogeno per la determinazione del contenuto di ferro e della capacita' ferrolegante di liquidi biologici
IT23396A/85 1985-12-27

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EP (1) EP0228060A1 (fr)
IT (1) IT1201512B (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011523A2 (fr) * 1990-10-11 1992-07-09 Synermed, Inc. Reactif et procede pour le titrage du fer dans le serum
US5186894A (en) * 1990-10-20 1993-02-16 Fuji Photo Film Co, Ltd. Dry analysis element for the analysis of iron ions
US5219760A (en) * 1988-05-26 1993-06-15 Boehringer Mannheim Gmbh Process for the determination of iron
US6627448B1 (en) * 1999-10-04 2003-09-30 Reference Diagnostics, Inc. Analyte-binding assay
US20090068750A1 (en) * 2005-05-12 2009-03-12 Wako Pure Chemical Industries, Ltd. Method of Determining Iron Concentration
US20110135054A1 (en) * 2002-08-21 2011-06-09 Gregerson Eugene A Apparatus and method for reconstruction of volumetric images in a divergent scanning computed tomography system

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1219848B (it) * 1988-03-03 1990-05-24 Miles Italiana Composti indicatori,metodo per la loro preparazione e loro impiego in un sistema di saggio della sideremia
DE10044639A1 (de) * 2000-09-08 2002-03-21 Teraklin Ag Indirektes Verfahren zur quantitativen Bestimmung der freien Bindungskapazität in einer wäßrigen Proteinlösung

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3506404A (en) * 1967-12-19 1970-04-14 Hoffmann La Roche Colorimetric method for determining iron in blood
US3537822A (en) * 1969-03-28 1970-11-03 American Hospital Supply Corp Method for the determination of serum iron and total iron binding capacity
US3667915A (en) * 1970-04-28 1972-06-06 Hoffmann La Roche Colorimetric methods and compositions for determining iron in blood
US3898042A (en) * 1974-01-02 1975-08-05 Dow Chemical Co Method and apparatus for continuously determining total copper in an aqueous stream
US4092117A (en) * 1975-08-13 1978-05-30 Inveresk Research International Device and method for monitoring the metal content of aqueous systems
US4224034A (en) * 1978-09-07 1980-09-23 American Monitor Corporation Assay of iron and iron binding protein reagents and methods
US4308027A (en) * 1978-11-08 1981-12-29 R.C.C. Societa' Ricerche Di Chimica Clinica S.R.L. Method and composition for direct determination of iron in blood serum
EP0061793A1 (fr) * 1981-04-01 1982-10-06 CHEMICAL LABORATORIES S.r.l. Procédé de préparation d'un réactif chromogénique pour la détermination de la concentration du fer dans le sérum et de la capacité de liaision de celui-ci avec le fer, et le réactif chromogénique ainsi obtenu
US4407962A (en) * 1980-02-26 1983-10-04 Istituto Sieroteropico E. Vaccinogeno Toscano "Sclavo" S.p.A. Composition for the colorimetric determination of metals
EP0137400A2 (fr) * 1983-09-26 1985-04-17 Wako Pure Chemical Industries, Ltd. Détermination de la capacité insaturée de fixation du fer
US4567150A (en) * 1981-11-20 1986-01-28 Sclavo, S.P.A. Method for determining transferrin and composition therefor
JPH05113039A (ja) * 1991-10-22 1993-05-07 Sekisui Chem Co Ltd 家屋ユニツトの据付け調整治具

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3506404A (en) * 1967-12-19 1970-04-14 Hoffmann La Roche Colorimetric method for determining iron in blood
US3537822A (en) * 1969-03-28 1970-11-03 American Hospital Supply Corp Method for the determination of serum iron and total iron binding capacity
US3667915A (en) * 1970-04-28 1972-06-06 Hoffmann La Roche Colorimetric methods and compositions for determining iron in blood
US3898042A (en) * 1974-01-02 1975-08-05 Dow Chemical Co Method and apparatus for continuously determining total copper in an aqueous stream
US4092117A (en) * 1975-08-13 1978-05-30 Inveresk Research International Device and method for monitoring the metal content of aqueous systems
US4224034A (en) * 1978-09-07 1980-09-23 American Monitor Corporation Assay of iron and iron binding protein reagents and methods
US4308027A (en) * 1978-11-08 1981-12-29 R.C.C. Societa' Ricerche Di Chimica Clinica S.R.L. Method and composition for direct determination of iron in blood serum
US4407962A (en) * 1980-02-26 1983-10-04 Istituto Sieroteropico E. Vaccinogeno Toscano "Sclavo" S.p.A. Composition for the colorimetric determination of metals
EP0061793A1 (fr) * 1981-04-01 1982-10-06 CHEMICAL LABORATORIES S.r.l. Procédé de préparation d'un réactif chromogénique pour la détermination de la concentration du fer dans le sérum et de la capacité de liaision de celui-ci avec le fer, et le réactif chromogénique ainsi obtenu
US4567150A (en) * 1981-11-20 1986-01-28 Sclavo, S.P.A. Method for determining transferrin and composition therefor
EP0137400A2 (fr) * 1983-09-26 1985-04-17 Wako Pure Chemical Industries, Ltd. Détermination de la capacité insaturée de fixation du fer
US4588695A (en) * 1983-09-26 1986-05-13 Wako Pure Chemical Industries, Ltd. Determination of unsaturated iron-binding capacity
JPH05113039A (ja) * 1991-10-22 1993-05-07 Sekisui Chem Co Ltd 家屋ユニツトの据付け調整治具

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Stephens et al., "Amer. Chem. Soc., Book and J. Div.", vol. 46, No. 6, pp. 692-696, May 1974.
Stephens et al., Amer. Chem. Soc., Book and J. Div. , vol. 46, No. 6, pp. 692 696, May 1974. *
Tabacco et al., "Clin. Chim. Acta", 114; pp. 287-290, 1981.
Tabacco et al., Clin. Chim. Acta , 114; pp. 287 290, 1981. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5219760A (en) * 1988-05-26 1993-06-15 Boehringer Mannheim Gmbh Process for the determination of iron
WO1992011523A2 (fr) * 1990-10-11 1992-07-09 Synermed, Inc. Reactif et procede pour le titrage du fer dans le serum
WO1992011523A3 (fr) * 1990-10-11 1992-09-17 Synermed Inc Reactif et procede pour le titrage du fer dans le serum
US5151370A (en) * 1990-10-11 1992-09-29 Synermed, Inc. Reagent and method for serum iron assay
US5186894A (en) * 1990-10-20 1993-02-16 Fuji Photo Film Co, Ltd. Dry analysis element for the analysis of iron ions
US6627448B1 (en) * 1999-10-04 2003-09-30 Reference Diagnostics, Inc. Analyte-binding assay
US20110135054A1 (en) * 2002-08-21 2011-06-09 Gregerson Eugene A Apparatus and method for reconstruction of volumetric images in a divergent scanning computed tomography system
US20090068750A1 (en) * 2005-05-12 2009-03-12 Wako Pure Chemical Industries, Ltd. Method of Determining Iron Concentration
US7785892B2 (en) * 2005-05-12 2010-08-31 Wako Pure Chemical Industries, Ltd. Method of determining iron concentration

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Publication number Publication date
IT1201512B (it) 1989-02-02
EP0228060A1 (fr) 1987-07-08
IT8523396A0 (it) 1985-12-27

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